G cmyc as Target for Anticancer Therapy

c-myc is one of the most frequently mutated genes in tumors and deregulation of c-myc is found in many, if not most, human tumors (Nesbit et al., 1999). In most cases the expression of c-myc is deregulated whereas mutations in the c-Myc protein are rare. Such enhanced and/or constitutive c-myc expression can be the result of mutations in the c-myc locus (e.g., chromosomal translocations, gene amplifications, proviral insertions, retroviral transductions) or in the signal transduction pathways that regulate c-myc expression (Marcu et al., 1992; Spencer and Groudine, 1991). Since in normal cells c-Myc induces apoptosis in the absence of sufficient amounts of survival factors (Askew et al., 1991; Evan et al., 1992) activation of the oncogene c-Myc strongly selects for a second mutation that eliminates an apoptosis pathway (e.g., p53) or for activation of a second cooperating oncogene that inhibits apoptosis and stimulates cell survival (e.g., Bcl-2, Bcl-xL, Ras) (Nilsson and Cleveland, 2003; Oster et al., 2002). However, if c-Myc-induced apoptosis is suppressed (e.g., by coexpression of the anti-apoptotic proteins Bcl-xL or Bcl-2 or by an excess of local survival factors) c-Myc activation alone is sufficient to trigger immediate carcinogenic progression in the absence of other cooperating oncogenic lesions (Luo et al., 2005; Pelengaris and Khan, 2003a; Pelengaris et al., 1999, 2002a,b). Vice versa, inactivation of the oncogene c-Myc alone can induce sustained tumor regression, reverse and revoke tumorigenesis and lead to the complete permanent loss of the neoplastic phenotype (Arvanitis and Felsher, 2005; Felsher, 2003, 2004; Giuriato and Felsher, 2003; Giuriato et al., 2004; Pelengaris and Khan, 2003a; Pelengaris et al., 2002a; Shachaf and Felsher 2005a,b):

In general, c-Myc inactivation in c-Myc-induced tumors results in proliferative arrest, re-differentiation, apoptosis, or/and vascular degeneration. Nevertheless, the outcome of c-Myc inactivation varies in different c-Myc-induced tumors depending on cell type, context, genetic, and epigenetic features. Thus, even brief c-Myc inactivation resulted in sustained tumor regression in osteogenic sarcoma (Jain et al., 2002), whereas prolonged c-Myc inactivation failed to cause tumor regression in the majority of mammary adenocarcinomas (Boxer et al., 2004; D'Cruz et al., 2001). Generally, four different outcomes of c-Myc inactivation can be distinguished in c-Myc-induced tumors. (1) Tumor regression with initial differentiation of tumor cells followed by their complete permanent elimination through apoptosis (lymphoma, leukemia: Felsher and Bishop, 1999a; Karlsson et al., 2003a). (2) Tumor regression with differentiation into normal mature quiescent tissue (osteogenic sarcoma: Jain et al., 2002; skin papilloma: Pelengaris et al., 1999; pancreatic islet /-cell carcinoma: Pelengaris et al., 2002b) where the re-differentiated cells are permanently refractory to subsequent reactivation of c-Myc, which either results in apoptosis (osteogenic sarcoma: Jain et al., 2002) or has no effect (skin papilloma: Flores et al., 2004). (3) Tumor regression with differentiation (in part accompanied by apoptosis) where the re-differentiated normal-appearing cells remain in a reversible state of tumor dormancy so that subsequent c-Myc reactivation immediately restores tumorigenesis (hepatocellular carcinoma: Shachaf et al., 2004; skin papilloma, pancreatic islet /-cell carcinoma (expressing Bcl-xL): Pelengaris et al., 2004).

(4) A failure to regress of the majority of mammary adenocarcinomas because they have become c-Myc-independent (in nearly 70% of cases due to activating point mutations in K-Ras2) coupled with spontaneous relapse of the majority of those breast tumors that fully regressed because they escape c-Myc-dependence through acquisition of additional genetic alterations (in a quarter of cases activating point mutations in K-Ras2) (Boxer et al., 2004; D'Cruz et al., 2001). Most if not all animals bearing fully regressed mammary adenocarcinomas harbor residual neoplastic cells, in which subsequent c-Myc reactivation results in rapid and full restoration of tumorigenesis (Boxer et al., 2004). This worse outcome of hepatocellular carcinoma, which are generally refractory to clinical treatment, pancreatic /-cell carcinoma, skin papilloma and especially mammary adenocarcinomas is at least in part due to their epithelial origin (Felsher, 2006). The high frequency of c-Myc-independence in the latter one is caused at least partially by their tendency to acquire activating K-Ras2 (and N-Ras) mutations, apparently often already during c-Myc-induced breast tumor initiation (Boxer et al., 2004; D'Cruz et al., 2001). Some hematopoietic tumors were also reported to relapse because of novel chromosomal translocations that rendered them c-Myc independent (Karlsson et al., 2003a). The acquisition of such new genetic alterations allowing tumor cells to escape c-Myc-dependence and thus to relapse may be promoted by c-Myc itself because it can cause genomic instability (Felsher and Bishop, 1999b; Karlsson et al., 2003b; Mai and Mushinski, 2003; Soucek and Evan, 2002).

Since c-Myc overexpression is causative of full tumorigenesis whereas interference with c-Myc expression is effective in tumor treatment c-Myc represents an attractive target for anticancer therapy (Hermeking, 2003; Oster et al., 2002; Ponzielli et al., 2005; Vita and Henriksson, 2006). Besides strategies to interfere with c-myc mRNA and c-Myc protein or to exploit c-Myc activation (Felsher and Bradon, 2003; Oster et al., 2002; Pelengaris and Khan, 2003b; Prochownik, 2004) several strategies to interfere with c-myc promoter activation are under intensive study (Ponzielli et al., 2005): DNA-binding antibiotics (Portugal, 2003; Snyder et al., 1991; Vaquero and Portugal, 1998), TFO (triplex-forming oligonucleotides) (Carbone et al., 2004a; Napoli et al., 2006; and references therein), small molecule ligands directed to block the DBD (FBP: Huth et al., 2004) and decoy DNAs (Seki et al., 2006) prevent transcription factor binding to the c-myc promoter. Small-molecule compounds that eliminate the interaction of /-catenin and TCF-4 (Lepourcelet et al., 2004) as well as a peptide aptamer that interrupts the interaction of Smad4 and LEF-1 (Lim and Hoffmann, 2006) inhibit the c-Myc protein or mRNA expression, respectively. Cationic porphyrins are used and c-myc-specific (expanded) cationic porphyrins are designed to sequester the NHE in inhibitory paranemic DNA structures (G-quadruplex:

Grand et al., 2002, 2004; Phan et al., 2005; Seenisamy et al., 2004, 2005; Siddiqui-Jain et al., 2002). Prominent examples of antibodies directed against components of signal transduction chains and other kinase inhibitors (Sawyers, 2003, 2004) successfully used in clinical anticancer therapy are Herceptin (Trastuzumab), a humanized monoclonal antibody specific for the transmembrane tyrosine kinase receptor HER-2/neu (hnRNP K; Emens, 2005; Nahta and Esteva, 2006), and Gleevec (Imatinib, STI-571), a potent small-molecule tyrosine kinase inhibitor with relative selective activity against ABL proto-oncogene, BCR-ABL fusion protein, PDGFR and c-Kit receptor (Jones and Judson, 2005; Peggs, 2004). Inhibition of expression (ETS-2: Carbone et al., 2004b) or function (NF-kB: Kanda et al., 2000) of transcription factors (Darnell, 2002) that activate the c-myc promoter is interesting as they often regulate also antiapoptotic or/and other proliferation genes. A detailed understanding of c-myc promoter regulation is one essential prerequisite to benefit from progress in diagnosis of individual genetic lesions in individual tumors and in target-directed drug design.

  2. Feedback Loops

The c-myc promoter responds to numerous signals and integrates these diverse and dynamic inputs to set the c-myc mRNA output (Chung and Levens, 2005; Liu and Levens, 2006). Then the transcription factor c-Myc mediates specific gene expression programs that relate to cell cycle progression and cell growth (Eisenman, 2001a,b; Grandori etal., 2000; Oster etal., 2002). Thereby it seems likely that c-myc transcription will respond to feedback from most (if not all) subsystems regulated by c-Myc (Levens, 2002, 2003). Fig. 8 shows the feedback coupling of the c-myc promoter to c-Myc regulated genes that are either direct c-Myc target genes or may be indirectly regulated by c-Myc [The Myc Target Gene Database (http://www.myccancergene.org/site/ mycTargetDB.asp); Adhikary and Eilers, 2005; Basso et al., 2006; Claassen and Hann, 1999; Coller et al., 2000; Dang, 1999; Eisenman, 2001a; Facchini and Penn, 1998; Fernandez et al., 2003; Frank et al., 2001; Frye et al., 2003; Grandori et al., 2000; Guo et al., 2000; Huang et al., 2003; Lee and Dang, 2006; Li et al., 2003; Luoro et al., 2002; Mao et al., 2003; Menssen and Hermeking, 2002; Nasi et al., 2001; Neiman et al., 2001; Nesbit et al., 2000; O'Connell et al., 2003; O'Hagan et al., 2000; Oster et al., 2002; Schuhmacher et al., 2001; Schuldiner et al., 2002; Toyo-Oka et al., 2006; Watson et al., 2002; Yu et al., 2002; Zeller et al., 2003, 2006].

Four different groups of feedback mechanisms can be distinguished (Fig. 8):

  1. Factors, which activate the c-myc promoter and whose expression is activated by c-Myc: In this group are prominent proliferation genes and notably many factors that activate E2F-dependent transcription. Since these factors and c-Myc will reciprocally enhance their expression they lead to a self-reinforcing transcription cycle, which drives cells through G1-phase and induces S-phase entry. Such a positive feedback program is important to efficiently promote cell proliferation and to provide robustness against competing antiproliferative signals.
  2. Factors, which repress the c-myc promoter and whose expression is repressed by c-Myc: In this group are prominent antiproliferation and differentiation genes, which act as antagonists to c-Myc in the proliferation/differentiation or proliferation/quiescence switch. In accordance, these factors and c-Myc reciprocally repress their expression. These antagonistic transcription programs are essential for cell fate determination. c-Myc itself represses the c-myc promoter. This negative feedback regulation of c-Myc autosuppression is important to ensure normal tissue homeostasis (see Section III.E; Facchini and Penn, 1998; Facchini et a/., 1994,1997; Grignani et a/., 1990; Penn et a/., 1990a; Potter and Marcu, 1997).
  3. Factors, which activate the c-myc promoter and whose expression is repressed by c-Myc: In this group are prominent proliferation genes. They induce c-myc expression and then repression of their expression by c-Myc serves to curtail the proliferation stimulus (Oster eta/., 2000). These negative feedback loops contribute to tight control of cell cycle progression (Facchini and Penn, 1998). This mechanism limits the time window of a particular mitogenic response and serves to maintain normal tissue homeostasis.
  4. Factors, which repress the c-myc promoter and whose expression is activated by c-Myc: In this group are prominent antiproliferation genes and tumor suppressors. c-Myc activates their expression and then they repress c-myc expression. This feedback control represents a security mechanism against inappropriate hyperproliferative signaling by c-Myc. A well characterized example is p53. In the absence of sufficient amounts of survival factors, high c-Myc levels cause p53-dependent and p53-independent apo-ptosis (Askew et a/., 1991; Evan et a/., 1992; Nilsson and Cleveland, 2003; Prendergast, 1999).

There exist also many other feedback mechanisms that are not shown in Fig. 8. For example, the Ras/ERK and PI3K/Akt pathways increase c-Myc protein stability (see Section IV.E; Sears et a/., 1999, 2000; Gregory and Hann, 2000; Gregory et a/., 2003; Sears, 2004; Sears and Nevins, 2002), c-Myc blocks the transactivation function of C/EBPa (Constance et a/., 1996; Mink et a/., 1996) and the c-Myc target gene eIF-4E (Bush et a/., 1998; Jones et a/., 1996; Rosenwald et a/., 1993) enhances c-myc translation (see Section IV.E; Clemens, 2004; De Benedetti and Graff, 2004; Massague, 2004; Rosenwald, 2004).

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